I. Infectious Bovine Rhinotracheitis
Bovine herpesvirus type 1 (hereinafter "BHV-1") more commonly known as infectious bovine rhinotracheitis virus (hereinafter "IBRV"), has been associated with respiratory, reproductive, enteric, ocular, central nervous system neonatal mammary, and dermal infections of cattle (Gibbs, E. P. J. et al, Vet. Bull, (London), 47:317-313 (1977)). Evidence for the association of IBRV with diseases of the respiratory tract was first obtained in the early 1950's. It has since become apparent that infectious bovine rhinotracheitis (hereinafter "IBR") has a worldwide distribution. By the mid-1960's, respiratory disease caused by IBRV resulted in losses in the United States estimated at about $25 million. Additional losses associated with IBRV infections have been due to abortion storms, dramatic losses in milk yield, metritis, enteritis, and meningitis, Since 1972, more severe forms of IBRV respiratory infections have become widespread in Canada and Western Europe. New IBR outbreaks probably result from exposure to imported asymptomatic IBRV carriers or exposure to infected animals prior to the onset of clinical disease. Hence, the importation of IBRV-infected livestock may be restricted or forbidden by some countries.
The spread of IBR in naturally and artificially bred cattle also poses a serious problem, especially with the continued, widespread use of frozen semen. In addition, recurrent shedding of IBRV from infected bulls constitutes a significant threat to the artificial insemination industry in the United States and to the worldwide distribution of bovine germ plasm. The incrimination of IBRV as the etiologic agent of oophoritis and salpingitis with resultant infertility and sterility adds to the seriousness of IBRV infections.
Natural IBRV infections of species other than cattle occur in swine, goats, water buffalo, wildebeasts, ferrets, and mink. Experimental infections have been established in swine, goats, mule deer, ferrets and rabbits (Joo, H. S. et al. Am. J. Vet. Med. Assoc., 45:1924-1927 (1984)).
The severity of illness resulting from IBRV infections depends upon the virus strain and on the age of the animal affected. After recovery from infection, animals may show clinical signs of recurrent disease without being reexposed to the virus. Recurrent disease without reexposure occurs because the virus remains dormant i e., latent, in neurons of the sensory ganglia of its host and can be reactivated, even after long periods (Rock, D. L. et al. J. Gen. Virol., 67:2515-2520 (1986)). Dexamethasone treatment can also provoke nasal shedding of the virus with or without clinical symptoms of active IBR. This suggests that reactivation and release from neuronal sites and, possible, persistent infection of other tissues can occur (Rossi, C. R. et al. Am. J. Vet. Res., 43:1440-1442 (1982)). Reactivation of IBRV from latency and symptomatic shedding can also occur spontaneously so that cattle latently infected with field strains of IBRV represent a sporadic source of virus transmission and herd infection.
II. Known IBR Vaccines
Control of IBR is based largely on vaccination. Currently, three types of IBR vaccines are being employed: (1) killed virus vaccines: (2) subunit vaccines;and (3) modified-live virus (hereinafter "MLV") vaccines (U.S Pat. Nos. 3,634,587; 3,925,544; and 4,291,019). Killed IBR vaccines are produced by treating the virus with chemicals, such as formalin or ethanol, and/or physical agents, such as heat or ultraviolet irradiation Subunit IBR vaccines are prepared by solubilizing IBRV-infected cell cultures with nonionic detergents and purifying some of the solubilized virus proteins (Babiuk, L. A. et al. Virol., 159:57-66 (1987)). Early MLV vaccines were designed for parenteral administration consisted of IBRV attenuated by rapid passage in bovine cell cultures. More recently, parenterally administered MLV vaccines have been attenuated by adaptation of IBRV to porcine or canine cell cultures, by adaptation to growth in cell culture at a low temperature (30.degree. C.) or by selection of heat-stable virus particles (56.degree. C. for 40 min). Specialized types of MLV vaccines are those administered intranasally. These MLV vaccines are attenuated by serial passage in rabbit cell cultures or by treatment of IBRV with nitrous acid followed by selection for temperature-sensitive mutants. A temperature-sensitive virus is one that replicates efficiently at 32.degree. C. to 38.degree. C., but not at about 39.degree. C. to 41.degree. C. (Todd, J. D. et al. J. Am. Vet. Med. Assoc., 159:1370-1374 (1971); Kahrs, R. F. et al. J. Am. Vet. Med. Assoc., 163:437-441 (1973); Smith, M. W. et al, Can. Vet. J., 19:63-71 (1978); Zygraich, N. et al, Res. Vet. Sci., 16:328-335 (1974); and U.S. Pat. Nos. 3,907,986 and 4,132,775).
The currently available IBR vaccines discussed above have serious disadvantages and have, therefore, proved unsatisfactory in commercial use. More specifically, although killed IBR vaccines are considered by some to be safer than MLV vaccines, i.e., they cannot establish latency and they eliminate the problem of postvaccination shedding, they are expensive to produce, must be administered several times, and disadvantageously require adjuvants. In addition, with their use, there is the possibility of fatal hypersensitivity reactions and nonfatal urticaria. Further, some infectious virus particles may survive the killing process and thus cause disease. Moreover, cattle vaccinated with killed IBR vaccines can be infected at a later time with virulent virus and can shed virulent virus, thereby spreading infection in the herd (Frerichs, G. N. et al. Vet. Rec., 111:116-122 (1982); and Wiseman, A. et al. Vet. Rec., 104:535-536 (1979)). Thus, although killed IBR vaccines can provide some protection against IBR, they are generally inferior to MLV vaccines in providing long-term protection.
Subunit vaccines are often less toxic than killed virus vaccines, and may induce novel immunologic effects which can be of significant value. The technique for subunit vaccine preparation involves removal of capsid proteins, while leaving intact antigenic proteins that elicit protective immunity. This creates a potential for the development of serologic procedures to differentiate vaccinated from naturally infected animals. Further, subunit vaccines, though antigenic, do not contain live virus and, thus, cannot be transmitted to other animals, cause abortion, or establish latency (Lupton, H. W. et al. Am. J. Vet. Res., 41:383-390 (1980); and le Q. Darcel, C. et al. Can. J. Comp. Med., 45:87-91 (1981)). However, subunit vaccines, like killed vaccines, do not generally prevent infection and latency when cattle are subsequently exposed to virulent IBRV field strains. Other disadvantages of subunit vaccines are the high cost of purification and the requirement of several injections with adjuvant.
MLV IBR vaccines have the important advantage that they produce rapid protection and activate cell-mediated and humoral components of the immune system. In the case of intranasal vaccination, localized immune responses that suppress later replication of virulent IBRV in the respiratory tract contribute significantly to protection. The local immune responses include production of interferon and antibodies in nasal secretions (Kucera, C. J. et al, Am. J. Vet. Res., 39:607-610 (1978)). Extensive utilization of MLV IBR vaccines has reduced the frequency of occurrence of IBR. However, most of the available MLV IBR vaccines are not entirely satisfactory. More specifically, there is concern as to their safety, especially if the vaccine virus itself produces latency and may be shed and transmitted to susceptible cattle.
Maximal utilization of intramuscularly (hereinafter "IM") administered MLV IBR vaccines has been especially hampered by the hazards of vaccine-induced abortions. That is, abortion rates as high as 60% have been reported after IM injection of some MLV IBR vaccines (Kahrs, R. F., J. Am. Vet. Med. Assoc., 171:1055-1064 (1977); and Kendrick, J. W. et al. Am. J. Vet. Res., 28:1269-1282 (1967)). In addition, with the MLV IBR vaccines currently in use, there is the danger of reversion to virulence.
In a search for safer MLV IBR vaccines, specialized vaccines have been developed (Todd, J. D. et al. J. Am. Vet. Med. Assoc., 159:1370-1374 (1971); Kahrs, R. F. et al. J. Am. Vet. Med. Assoc., 163:427-441 (1973); Smith, M. W. et al. Can. Vet. J., 19:63-71 (1979); Zygraich, N. et al. Res. Vet. Sci., 16:328-335 (1974); and Kucera, C. J. et al. Am. J. Vet. Res., 39:607-610 (1978)). These vaccines have been found to be immunogenic and safe for intranasal (hereinafter "IN") inoculation to pregnant cattle and can prevent abortions in pregnant cows which have been challenge-exposed to virulent IBRV. However, they have a disadvantage in that they can only be administered by the IN route. This is because, when administered IN, one such IBR vaccine replicates to a limited extent at the lower temperature of the upper respiratory tract. However, when administered IM, the vaccine replicates poorly or not at all at normal body temperature (Zygraich, N. et al. Res. Vet. Sci., 16:328-335 (1974)). On the other hand, another IBR vaccine is insufficiently attenuated for IM administration to pregnant animals although safe when given IN (Todd, J. D., J. Am. Vet. Med. Assoc., 163:427-441 (1973)). Furthermore, some of the vaccine strains produce mild or moderate respiratory disease even after IN administration, and they do not prevent signs of IBR following field challenge exposure (Kahrs, R. F. et al. J. Am. Vet. Med. Assoc., 163:437-441 (1973)).
Accordingly, neither the IM-administered MLV IBR vaccines, which are unsafe for pregnant cows, nor the MLV IBR vaccines that must be administered IN, discussed above fit comfortably into many of the current management practices. That is, vaccination of large numbers of cattle by the IN route is inconvenient and potentially dangerous to animal handlers. In addition, screening to identify pregnant animals prior to immunization is often not desirable or cost effective.
III. Attenuated Properties of Thymidine Kinase-Negative Herpesvirus Mutants
Recently, temperature-resistant, thymidine kinase negative (hereinafter "tk.sup.- ") IBR vaccines derived from the thymidine kinase positive (hereinafter "tk.sup.+ "), i.e., wild-type, Los Angeles strain of IBRV (ATCC No. VR-188) have been developed which overcome many of the problems that have limited the use of currently available vaccines (Kit, S. et al. Virol., 130:381-389 (1983); Kit, S. et al. Arch. Virol., 86:63-83 (1985); Kit, S. et al, Vaccine, 4 55-61 (1986); and U.S. Pat. Nos. 4,569,840 and 4,703,011, which articles and patents are incorporated by reference herein in their entirety). These IBR vaccines consist of plaque-purified IBRV isolates that replicate equally well at either 39.1.degree. C. or 34.5.degree. C. in rabbit skin and bovine tracheal cells. Hence, they are designated "temperature-resistant". This is in contrast to those IBRV strains that are designated, "temperature-sensitive", that is, those used for the IN-administered vaccines, which replicate only about 10.sup.-3 to 10.sup.-7 as well at 39.1.degree. C. as at 34.5.degree. C. In addition to the ability to replicate equally well at 39.1.degree. C. or 34.5.degree. C., the tk.sup.- IBR vaccines lack the ability to produce a functional thymidine kinase enzyme (hereinafter "TK") in infected cells. In one vaccine, designated IBRV(B8-D53) (ATCC No. VR-2066) (U.S. Pat. No. 4,569,840), the failure to produce a functional TK results from a mutagen-induced mutation. With a second vaccine, designated IBRV(NG)dltk (ATCC No. VR-2112) (U.S. Pat. No. 4,703,011), the failure to produce a functional TK results from a deletion of about 400 base pairs (hereinafter "bp") from the coding sequences of the IBRV tk gene as well as the insertion, into the IBRV tk gene, of a 40 bp oligonucleotide with stop codons in all three reading frames. The two characteristics, i.e., temperature resistance and tk.sup.-, directly contribute to the superiority of these mutant IBRVs as vaccines.
While the temperature-resistance and tk.sup.- characteristics of the IBRV mutants discussed above greatly improve their safety and usefulness of vaccines, the dormancy feature of IBRV makes it difficult to effect eradication of IBR through the application of quarantine measures which are intended to prevent the spread of IBR by the isolation of IBRV-infected herds and the slaughter of IBRV-infected animals. That is, with existing MLV IBR vaccines, it is difficult to determine whether a specific animal, which does not show symptoms of illness, is a carrier of a dormant IBRV, since usage of most current vaccines masks infections. Hence, since animals which appear healthy may actually be carriers and, thus, spreaders of IBRV, it is important to be able, even after vaccination, to identify infected animals and herds so as to be able to apply quarantine measures. Embodiments of the present invention were developed to meet this need.
In addition, some countries require that imported livestock, whether for breeding, for stocking of farms, or for market, be tested and shown not to be carriers of IBRV, i.e., the animals cannot be imported unless they are seronegative for IBRV, With current killed and MLV IBR vaccines, a producer who elects to protect his animals from the diseases which accompany the stresses of shipping, or who is forced by the circumstances of IBRV infection in an endemic region to vaccinate susceptible animals, finds himself at a severe economic disadvantage, since vaccination of the stock will result in a positive serological test for IBRV. Revaccination to enhance protection will further increase IBRV antibody titers. As a result, the farmer's ability to export valuable livestock and to sell his stock at home is restricted and he is at a disadvantage whether he vaccinates or does not vaccinate. An IBR vaccine that can safely be administered, protects cattle from disease and dormant infections caused by field strains of IBRV, has a low or non-existent probability of reversion to virulence and, yet, does not produce a positive serological test for IBRV would allow exportation of livestock and vaccination programs to be pursued unhindered by the fear of quarantine. Such a producer could then minimize losses within his own herd, while animal health authorities could continue with their respective control measures. Embodiments of the present invention were also developed in order to meet these needs.
IV. The Genomes of IBRV
The genomes of IBRV strains consist of linear, double-stranded, noncircularly permuted DNA molecules, approximately 135-140 kilobases (hereinafter "kb") in size. Analyses of the genomes of IBRV by electron microscopy and with restriction nuclease enzymes have shown that they consist of a sequence of DNA, designated as the short unique (hereinafter "U.sub.S ") sequence, about 13 kb in size. The U.sub.S sequence is bracketed by inverted repeat and terminal repeat sequences (hereinafter "IR.sub.S " and "TR.sub.S," respectively), each about 11.5 kb in size. Another unique sequence, i.e., the long unique (hereinafter "U.sub.L ") sequence, which is about 100 kb in size, comprises the remainder of the DNA molecule (Hammerschmidt, W. et al. J. Virol., 58:43-49 (1986); Engels, M. et al. Virus Res., 6:57-73 (1986/87); and Mayfield, J. E. et al. J. Virol., 47:259-264 (1983)). This genome structure exemplifies a Class D herpesvirus and is also found in pseudorabies virus (hereinafter "PRV"), equine herpesvirus types 1 and 3, and varicella-zoster virus. A consequence of such a genome structure is the ability to invert U.sub.S relative to U.sub.L leading to two isomeric structures of the DNA molecule. Physical maps of the genomes of several IBRV strains have been established for restriction endonucleases HindIII, BamHI, HpaI, EcoRI, and BstEII. Specific differences in the restriction endonuclease patterns of IBRV strains have been reported. Analyses of the restriction endonuclease patterns of the DNAs of more than 100 IBRV strains has allowed three different groups to be distinguished, but these do not correlate with epidemiological features (Engels, M. et al. Virus Res., 6:57-73 (1986/1987)).
Although IBRV encodes 50 to 100 genes, few of the viral genes have been located on the physical map of the IBRV genome. The IBRV tk gene is located within the HindIII-A restriction fragment at about 0.47 map units as shown in FIG. 1 (also see FIG. 1 of U.S. Pat. No. 4,703,011). The IBRV gene encoding glycoprotein gI has also been mapped in the HindIII-A fragment, to the left of the IBRV tk gene, at approximately map units 0.405 to 0.432 (Lawrence, W. C. et al. J. Virol., 60:405-414 (1986)).
One aspect of the present invention entails identification of the map location of another IBRV glycoprotein gene, that is, IBRV gIII. The data presented herein shows that the IBRV gIII gene maps at about 0.11 to 0.12 map units in the HindIII-I and BamHI-E fragments of IBRV (see FIG. 1).
V. IBRV Glycoproteins
Like most herpesviruses studied to date, IBRV specifies more than 25 structural polypeptides (Misra, V. et al. J. Virol., 40:367-378 (1981); and Van Drunen-en Littel-van den Hurk, S. et al. J. Virol., 59:401-410 (1986)). Among these polypeptides, to date, 10 or more glycosylated species have been identified. The virus-specific glycoproteins have a pivotal role in host-virus relationships since they are incorporated into the plasma membrane of the host cell, and ultimately become constituents of the virion envelope. In the latter capacity, they have important roles in recognition, attachment, and penetration of the virus into susceptible cells, in syncytia formation, and in different responses of the bovine immune system to IBRV infection, such as virus neutralization and the immune destruction of infected cells.
Recent immunoprecipitation experiments with monospecific antisera and monoclonal antibodies have shown that the following three sets of coprecipitating glycoproteins; (1) 180 and 97 Kilodaltons (hereinafter "kD") molecular weight glycoproteins; (2) 150 kD and 77 kD molecular weight glycoproteins; and (3) 130 kD, 74 kD and 55 kD molecular weight glycoproteins; are the major components of the IBRV envelope (Marshall, R. L. et al. J. Virol., 57:745-753 (1986)). These glycoproteins are also found on the surface of IBRV-infected cells and react with neutralizing monospecific antisera and monoclonal antibodies. Analyses under nonreducing conditions have shown that the 74 kD and 55 kD molecular weight glycoproteins interact through disulfide bonds to form the 130 kD molecular weight glycoprotein, designated IBRV gI (Van Drunen Littel-van den Hurk, S. et al. J. Virol., 59:401-410 (1986)). Partial proteolysis studies have also shown that the 180 kD molecular weight glycoprotein is a dimeric form of the 97 kD molecular weight glycoprotein, designated IBRV gIII, and that the 150 kD molecular weight glycoprotein is a dimer of the 77 kD molecular weight glycoprotein, designated IBRV gIV, but that these dimers are not linked by disulfide bonds.
In addition, minor glycoproteins of about 115 kD, 64 kD, and 45 kD molecular weight, and a nonglycosylated protein of about 107 kD molecular weight are removed from purified IBRV particles by detergent treatment. The 115 kD molecular weight glycoprotein, designated IBRV gII, appears to be virus-specific, since it is precipitated by monoclonal antibodies to IBRV. However, the 64 kD and 45 kD molecular weight glycoproteins are not precipitated by anti-IBRV convalescent antisera. Antisera to the 64 kD molecular weight glycoprotein precipitates several polypeptides from uninfected cell lysates, suggesting that the 64 kD molecular weight glycoprotein and, perhaps, the 45 kD molecular weight glycoprotein are proteins of cellular origin associated with the IBRV virion envelope.
Antigenically distinct precursors to each of the IBRV glycoproteins or glycoprotein complexes have been identified by monoclonal antibodies. The precursors for IBRV gI have molecular weights of about 117 kD and 62 kD. The precursors for IBRV gII, IBRV gIII and IBRV gIV have molecular weights of about 100 kD, 69 kD and 63 kD, respectively. These precursors are sensitive to endo-.beta.-N-acetylglucosaminidase H treatment, indicating that they represent partially glycosylated, high mannose-type intermediate forms generated by cotranslational glycosylation of the primary, unglycosylated precursors of IBRV gI, IBRV gII, IBRV gIII and IBRV gIV, which had apparent molecular weights of about 105 kD, 90 kD, 61 kD and 58 kD, respectively (Van Drunen Littel-van den Hurk, S., et al. J. Virol., 59:401-410 (1986)).
It is now recognized that in herpesviruses, more than one glycoprotein elicits virus-neutralizing antibodies and cytotoxic lymphocytes which aid in preventing infection and in recovery from infection. For example, monospecific antisera against each of the herpes simplex virus type 1 (hereinafter "HSV-1") glycoproteins, gB gC, gD, and gE, is able to neutralize virus and mediate complement-dependent cytolysis of virus-infected cells (Norrild, B. et al, J. Virol., 32:741-748 (1979)). Similarly, monoclonal antibodies against glycoproteins gB, gC, gD, and gF of herpes simplex virus type 2 (hereinafter "HSV-2") mediate immune lysis (Balachandran, N. et al. Infect. Immun., 37:1132-1137 (1982)). In addition, neutralizing antibodies are produced against each of the PRV glycoproteins gII, g92, and gp50. Further, passive immunization of animals with monoclonal antibodies directed against either PRV gp50 or PRV g92 protects them from wild-type infections (Hampl, H. et al. J. Virol., 52:583-590 (1984); Ben Porat, T. et al. Virol., 154:325-334 (1986); and Wathen, L. M. K. et al. Virus Res., 4:19-29 (1985)). With regard to IBRV major glycoproteins, gI, gIII and gIV, induce high levels of neutralizing antibodies in cattle and participate in antibody-dependent cell cytotoxicity (Babiuk, L. A. et al. Virol., 159:57-66 (1987)).
To develop a vaccine with a serological marker that distinguishes vaccinated animals from animals infected with field strains, it was necessary in the present invention to identify an IBRV glycoprotein gene which is nonessential for virus replication.
The IBRV gI glycoprotein appears to correspond to the HSV-1 gB glycoprotein (Laurence, W. C. et al,
J. Virol., 60:405-414 (1986)) and the PRV gII glycoprotein (Mettenleiter, T. C. et al. Virol., 152:66-75 (1986)). Further, the PRV gII glycoprotein like the IBRV gI glycoprotein, consists of about 70 kD and 58 kD molecular weight polypeptides covalently linked via disulfide bonds. In addition, the PRV gII glycoprotein shares 50% amino acid homology with the aligned HSV-1 gB glycoprotein and monospecific antisera made against the PRV gII glycoprotein immunoprecipitates the HSV-1 gB glycoprotein from infected cells (Robbins, A. K. et al, J. Virol., 61:2691-2701 (1987)). However, there is a great deal of data indicating that the HSV-1 gB gene and PRV gII gene are essential for virus replication (Spear, P. G., In: The Heroesviruses, 3:315-356 (1985); Marlin, S. D. et al. J. Virol., 3:128-136 (1985); Lawrence, W. C. et al. J. Virol., 60:405-414 (1986); and Bzik, D. J. et al. Virol., 137:185-190 (1984)). Thus, the IBRV gene encoding the corresponding IBRV glycoprotein, i.e.. IBRV gI, does not appear to be a promising candidate as an IBRV serological marker.
Viable HSV-1 and HSV-2 mutants which fail to express the gC glycoprotein have been isolated (Holland, T. C. et al. J. Virol., 52:566-574 (1984); Draper, K. G. et al. J. Virol., 51:578-585 (1984); Homa, F. L. et al. J. Virol., 58:281-289 (1986); and Johnson, D. C. et al. J. Virol., 58:36-42 (1986)). Similarly, a PRV mutant, PRV(dlg92/dltk), with a deletion in the PRV g92 gene has been isolated (Kit, S. et al. Am. J. Vet. Res., 48:780-793 (1987); and U.S. patent application Ser. No. 823,439, filed Jan. 28, 1986). PRV(dlg92/dltk) does not express a functional TK or antigenic PRV g92 polypeptides, but replicates to titers of over 10.sup.8 p.f.u./ml in rabbit skin and swine testicle cells. Further, extracts from cells infected with PRV(dlg92/dltk) do not contain a protein precipitated by anti-g92 monoclonal antibodies and pigs vaccinated with PRV(dlg92/dltk) do not produce antibodies to PRV g92 polypeptides, but do produce antibodies to other polypeptides. In addition, safety and efficacy studies have demonstrated that PRV(dlg92/dltk) elicits an immune response in pigs and mice and protects them from lethal doses of virulent challenge strains of PRV (Kit, S. et al, "Genetically Engineered Pseudorabies Virus Vaccine With Deletions in Thymidine Kinase and Glycoprotein Genes," Vaccines 87, pages 345-349, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (1987)). Deletion of the PRV g92 gene does not reduce the ability of PRV(dlg92/dltk) to generate a protective immune response or virus-neutralizing antibody in swine or mice. However, because of the PRV g92 deletion, animals immunized with PRV(dlg92/dltk) can be distinguished serologically from those infected with field strains of PRV. Hence, PRV(dlg92/dltk) exemplifies a safe and efficacious vaccine (U.S. patent application Ser. No. 823,439, filed Jan. 28, 1986). Thus, it was postulated in the present invention, that a better candidate for a dispensable IBRV glycoprotein marker gene might be the IBRV gene which corresponds to the HSV gC gene and the PRV g92 gene.
Other glycoprotein genes which are nonessential for IBRV replication in cultured cells might also be useful as IBRV serological markers. Nonessential IBRV glycoprotein genes are probably located in the U.sub.S segment of the IBRV genome since the HSV-1 glycoprotein genes gE (U.sub.S 8), gG (U.sub.S 4), and gI (U.sub.S 7) map in the U.sub.S portion of the HSV-1 genome, and they are "dispensable" for virus replication, at least in some cell lines in tissue culture (Longnecker, R. et al, Science, 236:573-576 (1987)); and PRV glycoprotein genes, gI, gp63, and gX, are located in the U.sub.S segment of the PRV genome and they are also "dispensable" for virus replication in cultured cells (Petrovskis, E. A. et al. J. Virol., 60:1166-1169 (1986); Petrovskis, E. A. et al, Virol., 159:193-195 (1987) Mettenleiter, T. C. et al. J. Virol , 61:2764-2769 (1987); and Thomsen, D. R. et al. J. Virol., 61:229-232 (1987)).
In the present invention, it has been possible for the first time to identify and map the location, and to clone and sequence the IBRV gIII gene. Further, in the present invention, it has been first determined that the IBRV gIII gene is dispensable for virus replication in cultured cells. As a result, it has been possible for the first time in the present invention to genetically engineer mutations in the IBRV gIII gene so as to provide IBRVs wherein animals vaccinated with such, due to a deletion and/or insertion mutations in the IBRV gIII gene, fail to produce any antigenic polypeptides encoded by the IBRV gIII gene and cannot revert to the production of the IBRV gIII antigens. As a result, animals vaccinated with such can be distinguished from animals infected with field strains of IBRV so as to enable the eradication of IBR disease through the application of quarantine measures. Additionally, in the present invention, it has been possible for the first time to provide an IBR vaccine which is both distinguishable from field strains, as discussed above, and which is not only effective in controlling the spread of IBR disease, but wherein the animals vaccinated with such, due to mutations also in the IBRV tk gene, are less likely to become carriers of the vaccine virus and are unlikely to acquire a dormant infection with pathogenic field strains.